*Target (protein/gene name): S-adenosylhomocysteine hydrolase *NCBI Gene # or RefSeq#:5655685 (full gene) or 223365960 (Chain A) *Protein ID (NP or XP #) or Wolbachia#: XP_001686974.1; Gene symbol: LmjF36.3910 *Organism (including strain): Leishmania major strain Friedlin, chain A Etiologic Risk Group (see link below): 2 (http://www.absa.org/riskgroups/parasitessearch.php?genus=Leishmania&species=major)
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Initiated by the bite of a sandfly, leishmaniasis is a tropical disease characterized by skin sores and damage of internal organs (1). Although noticeable symptoms may not appear at first, after an incubation of several weeks or months cutaneous leishmaniasis can cause bumps on the skin (cutaneous leishmaniasis). The infection can then develop rapidly, turning into volcanic sores that are raised on the outside with a crater in the center (1). Typically, these sores do not cause severe pain. The more serious side of the infection (visceral leishmaniasis) includes the enlarging of certain organs, such as the liver and spleen, and can effect bone marrow. Low red blood cell count due to the disease can also cause anemia, which takes several months or years to develop (2).
Currently, leishmaniasis is categorized as a tropical disease that is lacking in effective control measures (3). The disease is a vector-borne, caused by the parasitic protozoan Leishmania major. The high reproductive potential of L. major allows it to recover quickly after being treated with pharmaceutical drugs, which hinders global eradication programs (3). The protozoa are transferred from the gut of the sandfly to the human host once the human is bitten. The chosen drug target, S-adenosylhomocysteine hydrolase, is important in the cysteine-methionine and selenoamino acid metabolic pathways in the L. major parasite (4).
Link to TDR Targets page (if present):http://tdrtargets.org/targets/view?gene_id=27503 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):http://www.ncbi.nlm.nih.gov/protein/3G1U_A Essentiality of this protein: Yes 1. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: significant gain of fitness in bloodstream forms (3 days); Source study: alsford 2. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: significant gain of fitness in bloodstream forms (6 days); Source study: alsford 3. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford 4. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford Complex of proteins?: 4 chains; Protein-ligand complex; http://www.thesgc.org/structures/3g1u Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8 (according to TDR Targets)
Image of protein (PyMol with features delineated and shown separately): Figure 2. 3G1U protein shown as cartoon colored by chain. Figure 3. 3G1U protein chains shown as sticks colored with carbons green, nitrogen blue, and oxygen red.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>3G1U:A|PDBID|CHAIN|SEQUENCE MADYKVKDISLAEWGRKAIELAENEMPGLMELRREYGPSQPLKGAKIAGCLHMTVQTAVLIETLKALGAELRWSSCNIFS
TQDNAAAAIAKTGVPVFAWKGETDEEYEWCIAQTVKGFSGDGLPNMILDDGGDLTNLVIDRYPELVPKIFGISEETTTGVKNLYKRLSKGNLPISAINVNDSVTKSKFD
NLYGCRESLVDGIKRATDVMIAGKTCCVCGYGDVGKGCAAALRAFGARVVVTEVDPINALQASMEGYQVALVEDVMADAHIFVTTTGNDDIITSDHFPHMRDDAIVCN
IGHFDTEIQVGWLEANAKEHVEIKPQVDRYTMENGRHIILLAKGRLVNLGCASGHPSFVMSNSFTNQVLAQIELWSNRDNGKYPRGDKAGVFFLPKALDEKVAALHL
AHVGAKLTKLTPKQAEYINCPVNGPFKPDHYRY
(*sequence for Chain A only) *length of your protein in Amino Acids: 437 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 47828.7 Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 52995
Abs 0.1% (=1 g/l) 1.108, assuming all pairs of Cys residues form cystines
Ext. coefficient 52370
Abs 0.1% (=1 g/l) 1.095, assuming all Cys residues are reduced
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
*NCBI Gene # or RefSeq#: 5655685 (full gene) or 223365960 (Chain A)
*Protein ID (NP or XP #) or Wolbachia#: XP_001686974.1; Gene symbol: LmjF36.3910
*Organism (including strain): Leishmania major strain Friedlin, chain A
Etiologic Risk Group (see link below): 2 (http://www.absa.org/riskgroups/parasitessearch.php?genus=Leishmania&species=major)
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Initiated by the bite of a sandfly, leishmaniasis is a tropical disease characterized by skin sores and damage of internal organs (1). Although noticeable symptoms may not appear at first, after an incubation of several weeks or months cutaneous leishmaniasis can cause bumps on the skin (cutaneous leishmaniasis). The infection can then develop rapidly, turning into volcanic sores that are raised on the outside with a crater in the center (1). Typically, these sores do not cause severe pain. The more serious side of the infection (visceral leishmaniasis) includes the enlarging of certain organs, such as the liver and spleen, and can effect bone marrow. Low red blood cell count due to the disease can also cause anemia, which takes several months or years to develop (2).
Currently, leishmaniasis is categorized as a tropical disease that is lacking in effective control measures (3). The disease is a vector-borne, caused by the parasitic protozoan Leishmania major. The high reproductive potential of L. major allows it to recover quickly after being treated with pharmaceutical drugs, which hinders global eradication programs (3). The protozoa are transferred from the gut of the sandfly to the human host once the human is bitten. The chosen drug target, S-adenosylhomocysteine hydrolase, is important in the cysteine-methionine and selenoamino acid metabolic pathways in the L. major parasite (4).
References:
1. http://www.cdc.gov/parasites/leishmaniasis/
2. http://www.infoplease.com/cig/dangerous-diseases-epidemics/leishmaniasis-sand-fly-bug.html
3. http://www.ncbi.nlm.nih.gov/books/NBK11753/
4. http://tdrtargets.org/targets/view?gene_id=27503
Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=27503
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/protein/3G1U_A
Essentiality of this protein: Yes
1. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: significant gain of fitness in bloodstream forms (3 days); Source study: alsford
2. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: significant gain of fitness in bloodstream forms (6 days); Source study: alsford
3. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford
4. Gene/Ortholog: Tb11.01.1350 (OG4_10673); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Complex of proteins?: 4 chains; Protein-ligand complex; http://www.thesgc.org/structures/3g1u
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8 (according to TDR Targets)
*EC#: 3.3.1.1
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.3.1.1
Figure1. Screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Assay Reagent/Substrate: S-adenosylhomocysteine (S-AdoHcy) with ThioGlo1
Assay Method: Fluorescent
Supplier: Sigma-Aldrich
Cost: $63.70 for 10mg of substrate.............$594 for 1 mg ThioGlo
CAS: Substrate 979-92-0/ThioGlo 168639-87-0
Article: http://www.sciencedirect.com/science/article/pii/S0003269712005167
Reagent links:
http://www.sigmaaldrich.com/catalog/product/sigma/a9384?lang=en®ion=US
http://www.sigmaaldrich.com/catalog/product/sigma/94024?lang=en®ion=US
Structure Available (PDB or Homology model)
PDB #: 3G1U
Current Inhibitors:
CHEMBL1095280, CHEMBL1098945, CHEMBL8771, CHEMBL155085, CHEMBL1097885
BindingDB.org Article ID: 50031613
http://www.bindingdb.org/jsp/dbsearch/PrimarySearch_pubmed.jsp?pubmed=50031613&pubmed_submit=TBD
Expression Information (has it been expressed in bacterial cells): Yes
"Compounds that specifically inhibit S-adenosylhomocysteine hydrolase (SAHH; EC 3.3.1.1) interfere with the proliferation of Plasmodium malarial parasites, but efforts to identify the enzyme directly in parasite extracts have been unsuccessful. Here we report genetic and biochemical evidence for the presence of a gene encoding P. falciparum SAHH. The gene is transcribed as a 2.8-kilobase mRNA in erythrocytic stage parasites. Analysis of the open reading frame predicts a 53.9-kDa protein having conserved regions thought to be involved in NAD binding. The cDNA sequence has been incorporated into an Escherichia coli expression construct to confirm the function of the sahh product."
Article: Plasmodium falciparum S-adenosylhomocysteine hydrolase. cDNA identification, predicted protein sequence, and expression in Escherichia coli.
http://www.jbc.org/content/269/23/16364.short
Purification Method:
"S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose."
http://www.biochemj.org/bj/193/bj1930503.htm
"SAH hydrolase was purified to apparent homogeneity from bovine kidney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatically inactive."
http://go.galegroup.com/ps/retrieve.do?sgHitCountType=None&sort=RELEVANCE&inPS=true&prodId=AONE&userGroupName=txshracd2598&tabID=T002&searchId=R1&resultListType=RESULT_LIST&contentSegment=&searchType=AdvancedSearchForm¤tPosition=1&contentSet=GALE%7CA209407238&&docId=GALE|A209407238&docType=GALE&role=
Image of protein (PyMol with features delineated and shown separately):
Figure 2. 3G1U protein shown as cartoon colored by chain.
Figure 3. 3G1U protein chains shown as sticks colored with carbons green, nitrogen blue, and oxygen red.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>3G1U:A|PDBID|CHAIN|SEQUENCE MADYKVKDISLAEWGRKAIELAENEMPGLMELRREYGPSQPLKGAKIAGCLHMTVQTAVLIETLKALGAELRWSSCNIFS
TQDNAAAAIAKTGVPVFAWKGETDEEYEWCIAQTVKGFSGDGLPNMILDDGGDLTNLVIDRYPELVPKIFGISEETTTGVKNLYKRLSKGNLPISAINVNDSVTKSKFD
NLYGCRESLVDGIKRATDVMIAGKTCCVCGYGDVGKGCAAALRAFGARVVVTEVDPINALQASMEGYQVALVEDVMADAHIFVTTTGNDDIITSDHFPHMRDDAIVCN
IGHFDTEIQVGWLEANAKEHVEIKPQVDRYTMENGRHIILLAKGRLVNLGCASGHPSFVMSNSFTNQVLAQIELWSNRDNGKYPRGDKAGVFFLPKALDEKVAALHL
AHVGAKLTKLTPKQAEYINCPVNGPFKPDHYRY
(*sequence for Chain A only)
*length of your protein in Amino Acids: 437 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 47828.7
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence (paste as text only):
atggcggact acaaggtaaa agacatcagc ctcgcggagt ggggccgcaa ggcgatcgag ctcgcggaaa acgagatgcc cggtctgatg gagctgcgcc gcgagtacgg accctcccag
ccgctgaagg gcgccaagat tgccggctgc ctgcacatga ccgtacagac tgccgtgctg atcgagaccc tcaaggccct cggtgcggag ctgcgctggt cgtcgtgcaa catcttttcc
actcaggata acgccgctgc ggccattgcc aagactggcg taccggtgtt tgcgtggaag ggtgagacag acgaggagta tgagtggtgc atcgcccaga cagtgaaggg cttcagcggt
gacggtctgc cgaacatgat cctcgacgac ggtggcgacc tcacgaatct ggtgatcgac cgctaccccg agctcgtgcc caagatcttc ggtatctccg aggagaccac gacgggtgtg
aagaacctgt acaagcgcct gagcaagggc aacctcccca tctccgccat caacgttaac gacagcgtga cgaagagcaa gttcgacaac ctttacggct gccgagagtc cttggtggac
ggcatcaagc gcgccacgga tgtcatgatt gccggcaaga cgtgctgcgt gtgcggatac ggtgatgttg gtaagggctg cgccgccgcc ctgcgcgcct tcggcgctcg cgttgtggta
acggaggtgg accctatcaa tgcccttcag gcctccatgg agggctacca ggtcgctctg gtcgaagacg tcatggccga tgcgcacatc ttcgtgacga ccaccggcaa cgatgacatc
atcacctctg accacttccc tcacatgcgc gacgacgcca ttgtgtgcaa catcggccac ttcgacacgg agatccaggt gggatggctt gaggcaaacg ccaaagagca cgtggaaatc
aagcctcagg tggaccgcta taccatggag aacggccgtc acatcatcct gctggctaag ggccgcctgg tcaacctcgg ctgcgccagc ggtcacccgt ccttcgtgat gtccaactcc
ttcacgaacc aggtgctggc tcagatcgag ctctggagca accgcgacaa cggcaagtac ccgcgcggtg acaaggccgg cgtgttcttc ctgcccaagg cgctcgatga gaaggtcgcc
gccctccacc tcgcccacgt cggcgccaag ctgacaaaac tgaccccgaa gcaggccgag tacatcaact gcccggtcaa cggcccgttc aagccggacc actaccgcta ctaa
http://www.ncbi.nlm.nih.gov/nuccore/157877302?from=1&to=1314&sat=4&sat_key=70152472
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**